The complete mitochondrial genome of a fouling mussel, Xenostrobus atratus (Mollusca: Mytilidae), and its phylogenetic implication

Abstract In this study, we report the female-lineage mitochondrial genome of Xenostrobus atratus for the first time. The circular mitochondrial genome is 14,806 bp in length and contains 12 protein-coding genes, 22 transfer RNA genes, and two ribosomal RNA genes. All genes are encoded on the heavy strand. The genome composition is A + T biased (66.6%), with 25.2% A, 41.4% T, 21.7% G and 11.7% C. A Bayesian inference (BI) phylogenetic tree was constructed based on the mitochondrial genomes of X. atratus and 46 other Mytilidae species. Our results demonstrate that X. atratus and Limnoperna fortunei have distinct lineages, opposing synonymizing Xenostrobus within Limnoperna. According to this study, the validity of the subfamily Limnoperninae and genus Xenostrobus is strongly supported. However, there is still an urgent need for more mitochondrial data to decide to which subfamily X. atratus belongs.


Introduction
Xenostrobus species occupys different habitats, such as tropical mangroves, rocky substrates upstream and downstream of estuaries, and rocky intertidal zone (Colgan and da Costa 2013). There are eight extant species of Xenostrobus (Colgan et al. 2020), and only one, Xenostrobus atratus, is reported for China (Bernard 1993;Wang 1997). Due to the similarity of morphological characteristics with an invasive freshwater mussel, Limnoperna fortunei (Beu 2006), X. atratus used to be named Limnoperna atrata. The relationship between the two genera is still in debate. For example, Beu (2006) placed the species within the Limnoperna genus, however, Colgan and da Costa (2013)-based on differences in BEAST-estimated age and cox1 amino acid sequences-objected to the suggestion that Xenostrobus and Limnoperna were congeneric. Therefore, convincing evidence is needed to consider the relationships between Xenostrobus and Limnoperna. In addition, the fouling property that affects the normal growth and development of mangroves (He, 2002) draws considerable attention to X. atratus. Since characteristics of mitochondrial genomes are efficient and powerful in phylogenetic studies in Mytilidae (Lee et al. 2019;Zhang et al. 2019), the mitochondrial genome of X. atratus was sequenced for the first time in this study. An interesting feature of Mytilidae mtDNA is Doubly Uniparental Inheritance (DUI), which has two types of mitochondrial DNA [female-lineage type (F-type) and male-lineage type (M-type)] and M-type mitogenome is only present in the gonads of male individuals. In mytlids, the F-type and M-type mitogenomes appear to exhibit an estimated 10% difference in base sequences (Fisher and Skibinski, 1990). DUI species were found not only in marine mussels but also in freshwater mussels (Soroka, 2020). There are more than 100 species of bivalve mollusks known to have DUI (Zouros, 2020).

Genome sequencing, assembly and annotation
The X. atratus DNA library was sequenced by Personalbio Biotechnology Co., Ltd (Shanghai, China) using an Illumina NovaSeq 6000 with an average insert size of 400 bp, which was constructed using TruSeq TM Nano DNA Sample Pre Kit. Approximately 3.98 GB of raw data for X. atratus were generated with 150 bp paired-end reads. Adapters were removed by Adapter Removal v.2 (Schubert et al. 2016). Clean data was assembled de novo using A5-miseq v20150522 (Coil et al. 2015) and SPAdesv3.9.0 (Bankevich et al. 2012). We determined the location of genes using the MITOS web server (http://mitos2.bioinf.uni-leipzig.de/index.py). The annotated sequence has been submitted to GenBank with Accession no. OM001008 (F type).

Phylogenetic analysis
A Bayesian inference (BI) phylogenetic tree was conducted using sequence data from the concatenated sets of 12 PCGs and 2 rRNAs of the X. atratus mitogenome and 46 Mytilidae species previously published, with Crassostrea gigas (Ostreidae) and Anadara sativa (Arcidae) as outgroups. The best substitution model for nucleotide sequences selected by Modelfinder (Kalyaanamoorthy et al. 2017) was GTR þ F þ G. BI tree was established using software MrBayes v.3.2.6 (Ronquist et al. 2012) , with 2,000,000 generations and discarding the first 25% as burnin.

Results
The mitochondrial genome of X. atratus is a closed-circular DNA molecule with a length of 14,806 bp, containing 12 protein-coding genes (PCGs), including 7 subunits of NADH dehydrogenase (nad1-6 and nad4l), 3 subunits of cytochrome c oxidase (cox1-3), one subunits of ATPase (atp6), cytochrome b (cytb), 22 transfer RNA genes (tRNAs) and 2 ribosomal RNA genes (12S rRNA and 16S rRNA). The total length of the noncoding regions is 449 bp, accounting for 3.03% of the mitochondrial genome. No atp8 gene was found by comparison with closely related species. Song et al. (2009) hypothesized that the absence of gene atp8 in some species resulted from adaptation to environmental conditions, but, this coding gene has been detected in many fresh-water and marine bivalves. This might be explained by the inherent structural properties (such as short length) and extreme variability of atp8 among bivalves that hider the proper detection and annotation of this gene (Breton et al. 2010;Gait an-Espitia et al. 2016).

Dicussion
The validity of the subfamily Limnoperninae has been doubted and the genus Limnoperna was sometimes placed under Arcuatulinae (Huber 2010). In this study, L. fortunei clusters with X. atratus, Bathymodiolinae and Modiolinae, constituting one of the two main clades in Mytilidae, and not with Arcuatulinae species. Consequently, it is more likely that Limnoperninae is valid. Lee et al. (2019) proposed that Limnoperninae is sister to (Bathymodiolinae þ Modiolinae) based on a mitochondrial genome phylogeny, but the newly sequenced species, X. atratus, takes its position and clusters with (Bathymodiolinae þ Modiolinae) in this study, and they are then sister to Limnoperninae (posterior probability ¼ 1). In particular, X. atratus and L. fortunei are closely related but have distinct lineages with long phylogenetic branch lengths, longer than most of the other closely related species within the Bathymodiolinae and Modiolinae, opposing the synonym of the two genera raised by Beu (2006). Additionally, since gene orders among low-level taxonomic species are highly conserved and have been proven to be an effective phylogenetic tool in Mollusca (Boore and Brown, 1998;Ghiselli et al. 2021), the very large difference in the PCG order and tRNA arrangement between X. atratus and L. fortunei provides further evidence for supporting the validity of genus Xenostrobus (Figure 1(b)). Nonetheless, it's still too early to decide which subfamily X. atratus belongs to based on current studies. Additional molecular data are needed to solve this controversy and deeper relationships among Mytilidae species.

Ethical approval
This study does not need ethical approval or permissions to collect, handling, and transport of the samples.

Author contributions
Houmei Li conducted the investigation, data curation, and drafting of the paper. Chenghua Li, Peizhen Ma, and Haiyan Wang contributed to the analysis and interpretation of data for the work. Zhen Zhang was involved in charge of the methodology and the final approval of the version to be published. All authors contributed to the article and approved the submitted version.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This work was supported by the National Key R&D Program of China [2019YFD0902100].

Data availability statement
The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at (https://www.ncbi.nlm.nih.gov/) under accession no.OM001008. The associated BioProject, SRA and Bio-Sample numbers are PRJNA808856, SRR18136193, and SAMN26116627 respectively.